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Results for “Drug Resistance, Multiple, Fungal”

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At least 19 recordsLinked to original sources

PubMed2002

Modulation by flavonoids of cell multidrug resistance mediated by P-glycoprotein and related ABC transporters.

Cancer cell resistance to chemotherapy is often mediated by overexpression of P-glycoprotein, a plasma membrane ABC (ATP-binding cassette) transporter which extrudes cytotoxic drugs at the expense of ATP hydrolysis. P-glycoprotein (ABCB1, according to the human gene nomenclature committee) consists of two homologous halves each containing a transmembrane domain (TMD) involved in drug binding and efflux, and a cytosolic nucleotide-binding domain (NBD) involved in ATP binding and hydrolysis, with an overall (TMD-NBD)2 domain topology. Homologous ABC multidrug transporters, from the same ABCB family, are found in many species such as Plasmodiumfalciparum and Leishmania spp. protozoa, where they induce resistance to antiparasitic drugs. In yeasts, some ABC transporters involved in resistance to fungicides, such as Saccharomyces cerevisiae Pdr5p and Snq2p, display a different (NBD-TMD)2 domain topology and are classified in another family, ABCG. Much effort has been spent to modulate multidrug resistance in the different species by using specific inhibitors, but generally with little success due to additional cellular targets and/or extrusion of the potential inhibitors. This review shows that due to similarities in function and maybe in three-dimensional organization of the different transporters, common potential modulators have been found. An in vitro 'rational screening' was performed among the large flavonoid family using a four-step procedure: (i) direct binding to purified recombinant cytosolic NBD and/or full-length transporter, (ii) inhibition of ATP hydrolysis and energy-dependent drug interaction with transporter-enriched membranes, (iii) inhibition of cell transporter activity monitored by flow cytometry and (iv) chemosensitization of cell growth. The results indicate that prenylated flavonoids bind with high affinity, and strongly inhibit drug interaction and nucleotide hydrolysis. As such, they constitute promising potential modulators of multidrug resistance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2005

Isolation, identification and antifungal susceptibility of lemon pathogenic and non pathogenic fungi.

Numerous species of filamentous fungi were isolated from lemon on different plantations in the province of Tucuman, Argentina. The techniques suggested by the Subcommittee of Antifungal Susceptibility of the National Committee for Clinical Laboratory Standards, (USA) were adapted. The effect of three different concentrations of the fungicides imazalil, guazatine, SOPP and thiabendazole on the fungi Fusarium oxysporum, Fusarium moniliforme, Aspergillus niger, Aspergillus flavus, Aspergillus clavatus, Geotrichum candidum, Rhizopus sp, Penicillium sp, Penicillium digitatum and Mucor sp were studied. All the tested strains were resistant to thiabendazole. We assayed a mixture of SOPP (5%), guazatine (350 ppm) and imazalil (100 ppm), which showed a synergic effect on Rhizopus sp. Mucor sp was the only fungus resistant to the four fungicides tested as well as to the above mentioned mixture.

Aspergillus↗

PubMed2006

Multiple cis-acting sequences mediate upregulation of the MDR1 efflux pump in a fluconazole-resistant clinical Candida albicans isolate.

Overexpression of the MDR1 gene, which encodes a multidrug efflux pump of the major facilitator superfamily, is a frequent cause of resistance to the antimycotic agent fluconazole and other metabolic inhibitors in clinical Candida albicans strains. Constitutive MDR1 overexpression in such strains is caused by mutations in as yet unknown trans-regulatory factors. In order to identify the cis-acting sequences in the MDR1 regulatory region that mediate constitutive MDR1 upregulation, we performed a promoter deletion analysis in the genetic background of an MDR1-overexpressing clinical C. albicans isolate. We found that several different regions in the MDR1 promoter can mediate MDR1 overexpression in this isolate. In contrast, deletion of one of these regions abolished benomyl-induced MDR1 expression in a C. albicans laboratory strain. These results suggest that multiple transcription factors control expression of the MDR1 efflux pump in C. albicans and that the mutation(s) that causes constitutive MDR1 overexpression and drug resistance in clinical C. albicans isolates affects the activities of several of these transcription factors.

Antifungal Agents↗

PubMed2000

Cross-talk between transcriptional regulators of multidrug resistance in Saccharomyces cerevisiae.

Multiple or pleiotropic drug resistance often arises in the yeast Saccharomyces cerevisiae due to genetic alterations of the functional state of the Cys(6)-Zn(II)(2) transcription factors Pdr1p and Pdr3p. Single amino acid substitutions give rise to hyperactive forms of these regulatory proteins, which in turn cause overproduction of downstream target genes that directly mediate multidrug resistance. Previous work has identified a novel Cys(6)-Zn(II)(2) transcription factor designated Yrr1p as mutant forms of this protein confer high level resistance to the cell cycle inhibitor reveromycin A and DNA damaging agent 4-nitroquinoline-N-oxide. In the present study, we demonstrate that Yrr1p also mediates oligomycin resistance through activation of the ATP-binding cassette transporter-encoding gene YOR1. Additionally, insertion of triplicated copies of the hemagglutinin epitope in the C-terminal region of Yrr1p causes the protein to behave as a hyperactive regulator of transcription. We have found that YRR1 expression is both controlled in a Pdr1p/Pdr3p-dependent manner and autoregulated. Chromatin immunoprecipitation experiments also show that Yrr1p associates with target promoters in vivo. Together these data argue that the signal generated by activation of Pdr1p and/or Pdr3p can be amplified through the action of these transcriptional regulatory proteins on downstream target genes, like YRR1, that also encode transcription factors.

ATP-Binding Cassette Transporters↗

PubMed2005

Aspergillus lentulus sp. nov., a new sibling species of A. fumigatus.

In a prior study, we identified seven clinical isolates of an Aspergillus sp. that were slow to sporulate in multiple media and demonstrated decreased in vitro susceptibilities to multiple antifungals, including amphotericin B, itraconazole, voriconazole, and caspofungin. These isolates were initially considered to be variants of Aspergillus fumigatus because of differences in mitochondrial cytochrome b sequences and unique randomly amplified polymorphic DNA PCR patterns (S. A. Balajee, M. Weaver, A. Imhof, J. Gribskov, and K. A. Marr, Antimicrob. Agents Chemother. 48: 1197-1203, 2004). The present study was performed to clarify the taxonomic status of these organisms by phylogenetic analyses based on multilocus sequence typing of five genes (the beta-tubulin gene, the rodlet A gene, the salt-responsive gene, the mitochondrial cytochrome b gene, and the internal transcribed spacer regions). Results revealed that four of the seven variant isolates clustered together in a clade very distant from A. fumigatus and distinct from other members of the A. fumigatus group. This new clade, consisting of four members, was monophyletic with strong bootstrap support when the protein-encoding regions were analyzed, indicating a new species status under the phylogenetic species concept. Phenotype studies revealed that the variant isolate has smaller conidial heads with diminutive vesicles compared to A. fumigatus and is not able to survive at 48 degrees C. Our findings suggest the presence of a previously unrecognized, potentially drug-resistant Aspergillus species that we designate A. lentulus.

Aspergillus↗

PubMed2006

Isolation and characterization of promoters suitable for a multidrug-resistant marker CuYAP1 in the yeast Candida utilis.

The overexpression of CuYAP1 by the CuGAP1 promoter (Pgap) was recently shown to function as a drug-resistant selection marker for the industrially important yeast Candida utilis. In order to increase the efficiency of selection, we screened for promoters better than Pgap to express CuYAP1. Two restriction fragments, P2-1-2 (0.5 kbp) and P2-33-2 (1.4 kbp), gave higher cycloheximide resistance, and five- to 10-fold of the transformants were selectable by using these fragments. These promoters were found to be at the 5' of the ribosomal protein genes, RPL31 and RPL29, respectively. Interestingly, their transcription activity was less than one-tenth that of Pgap in the absence of cycloheximide. The transcription also increased by the addition of blasticidin S or hygromycin B and heat shock. These novel characteristics will be suitable for an economical marker of the recombinant cell. The DDBJ/EMBL/GenBank Accession Nos. for P2-1, P2-33-2, RPL31 and RPL29 are AB206952, AB206953, AB208646 and AB208647, respectively.

Amino Acid Sequence↗

PubMed2002

Use of a YAP1 overexpression cassette conferring specific resistance to cerulenin and cycloheximide as an efficient selectable marker in the yeast Saccharomyces cerevisiae.

Drug-resistance markers for yeast transformation are useful because they can be applied to strains without auxotrophic mutations. However, they are susceptible to technical difficulties, namely lower transformation efficiency and the appearance of drug-resistant mutants without the marker. To avoid these problems, we have constructed a phosphoglycerate kinase (PGK) promoter-driven YAP1 expression cassette, called PGKp-YAP1. Yeast cells containing PGKp-YAP1 were resistant to cycloheximide, a protein synthesis inhibitor, and also to cerulenin, a fatty acid synthesis inhibitor, but not to other drugs tested. The transformation efficiency of PGKp-YAP1 using cerulenin selection was comparable to that using a URA3 auxotrophic marker when low concentrations of cerulenin were used. Non-transformed drug-resistant colonies did appear on the low-concentration cerulenin plates. However, these non-transformed colonies could easily be identified, based on their cycloheximide sensitivity and/or their resistance to aureobasidin A to which the transformants were sensitive. Therefore, the dual drug resistance of PGKp-YAP1 could be used as an effective selection for PGKp-YAP1 recipient cells. The PGKp-YAP1 marker was used to disrupt the LYS2 gene and to transform an industrial yeast strain, indicating that this marker can be used for efficient and reliable gene manipulations in any Saccharomyces cerevisiae strain.

Antifungal Agents↗

PubMed1994

Saccharomyces cerevisiae YDR1, which encodes a member of the ATP-binding cassette (ABC) superfamily, is required for multidrug resistance.

A multidrug resistance gene, YDR1, of Saccharomyces cerevisiae, which encodes a 170-kDa protein of a member of the ABC superfamily, was identified. Disruption of YDR1 resulted in hypersensitivity to cycloheximide, cerulenin, compactin, staurosporine and fluphenazine, indicating that YDR1 is an important determinant of cross resistance to apparently-unrelated drugs. The Ydr1 protein bears the highest similarity to the S. cerevisiae Snq2 protein required for resistance to the mutagen 4-NQO. The drug-specificity analysis of YDR1 and SNQ2 by gene disruption, and its phenotypic suppression by the overexpressed genes, revealed overlapping, yet distinct, specificities. YDR1 was responsible for cycloheximide, cerulenin and compactin resistance, whereas, SNQ2 was responsible for 4-NQO resistance. The two genes had overlapping specificities toward staurosporine and fluphenazine. The transcription of YDR1 and SNQ2 was induced by various drugs, both relevant and irrelevant to the resistance caused by the gene, suggesting that drug specificity can be mainly attributed to the functional difference of the putative transporters. The transcription of these genes was also increased by heat shock. The yeast drug-resistance system provides a novel model for mammalian multidrug resistance.

ATP-Binding Cassette Transporters↗

PubMed2002

A novel ABC transporter gene, PMR5, is involved in multidrug resistance in the phytopathogenic fungus Penicillium digitatum.

We have cloned a novel ABC transporter gene PMR5 from the phytopathogenic fungus Penicillium digitatum by RT-PCR using degenerate primers. The deduced amino acid sequence of PMR5 showed 37% identity to PMR1 from the same fungus, 71% identity to AtrB from Aspergillus nidulans, and 65% identity to BcatrB from Botrytis cinerea. Disruption mutants for PMR5 were generated in two independent P. digitatum strains and their phenotypes were characterized. These mutants displayed increased sensitivity to thiabendazole (a benzimidazole), benomyl (a benzimidazole), dithianon (a quinone), resveratrol (the phytoalexin of grape), and camptothecin (an alkaloid). Delta pmr1 disruption mutants were previously reported to show resistance to demethylation inhibitors (DMIs). These mutants were found also to display increased sensitivity to phloretin (the phytoanticipin of apples), camptothecin and oligomycin (an antibiotic). Transcription of PMR1 and PMR5 was strongly induced in response to several toxicants, including DMIs that specifically induced PMR1. In contrast, dithianon and resveratrol specifically induced PMR5 transcription. These findings indicate that expression of the two ABC transporter genes is regulated differently, and that they have complementary roles in multidrug resistance, with each having different substrate-specificities.

ATP-Binding Cassette Transporters↗

PubMed2005

Susceptibilities to amphotericin B and fluconazole of Candida species in TSARY 2002.

Susceptibilities to amphotericin B and fluconazole of 909 Candida species collected during the Taiwan Surveillance of Antimicrobial Resistance of Yeasts (TSARY) in 2002 were determined by the broth microdilution method. There were 395 (43.5%) Candida albicans, 244 (26.8%) C. tropicalis, 187 (20.6%) C. glabrata, 63 (6.9%) C. parapsilosis, 9 (1%) C. krusei, and 11 (1.2%) others. Among them, 23 (2.5%) isolates were resistant to amphotericin B. They consisted of 10 C. glabrata, 6 C. krusei, 3 C. albicans, 1 C. tropicalis, 1 C. parapsilosis, and 2 others. The resistance rate to amphotericin B has increased compared with that of TSARY 1999 (2.5% versus 0.5%). There were 7 C. krusei, 5 C. albicans, 3 C. glabrata, and 2 others isolates resistant to fluconazole. The resistance rate to fluconazole has decreased from 8.4% in 1999 to 1.9% in 2002. A pattern of coresistance to both amphotericin B and fluconazole was observed.

Amphotericin B↗

PubMed2002

Antifungal drug resistance of pathogenic fungi.

Pathogenic fungi have many complex mechanisms of resistance to antifungal drugs. Information about the clinical, cellular, and molecular factors contributing to antifungal-drug resistance continues to accumulate. We critically review the diagnosis, epidemiology, and mechanisms of antifungal drug resistance of pathogenic fungi. Better understanding of this resistance should assist in developing better detection strategies for preventing and treating refractory mycoses in the future.

Antifungal Agents↗

PubMed2002

Antifungal activities of fluconazole, caspofungin (MK0991), and anidulafungin (LY 303366) alone and in combination against Candida spp. and Crytococcus neoformans via time-kill methods.

The activities of the echinocandins caspofungin and anidulafungin were evaluated alone and in combination with fluconazole using time-kill methods against isolates of Candida albicans, Candida glabrata, Candida tropicalis, Candida krusei, and Cryptococcus neoformans. Antifungal concentrations tested against each isolate were 0.5 microg/mL and 20 microg/mL of fluconazole and 0.007 microg/mL and 2 microg/mL of both caspofungin and anidulafungin. In addition, 20 microg/mL of fluconazole was tested with 2 microg/mL of caspofungin and anidulafungin to test for additive or antagonistic activity. Finally 0.5 microg/mL of fluconazole was tested with 0.007 microg/mL of caspofungin and anidulafungin to test for synergy. Combinations of fluconazole and caspofungin or anidulafungin resulted in indifference. Azole-echinocandin combinations do not produce antagonistic effects; therefore, combinations of these agents may warrant future clinical evaluation.

Anidulafungin↗

PubMed2005

In vitro activities of voriconazole, fluconazole, itraconazole and amphotericin B against non Candida albicans yeast isolates.

Antifungal susceptibility testing was performed on 197 yeast isolates from the BCCM/IHEM biomedical fungi and yeasts collection (Belgian Co-ordinated Collections of Micro-organisms / IPH-Mycology) to study the in vitro activity of voriconazole against fluconazole, itraconazole and amphotericin B. MICs of the four antifungal agents were determined by an adapted NCCLS M27-A microdilution reference method. MIC readings were visually and spectrophotometrically determined. Optical density data were used for calculation of the MIC endpoints. For amphotericin B, the MIC endpoint was defined as the minimal antifungal concentration that exerts 90% inhibition, compared to the control growth. The azoles endpoints were determined at 50% inhibition of growth. The MIC distribution of voriconazole susceptibilities showed that 193 isolates had a MIC < or = 2 microg/ml and 185 a MIC < or = 1 microg/ml. Cross-tabulation of voriconazole, fluconazole, and itraconazole MICs indicated that voriconazole MICs raised with fluconazole and itraconazole MICs. The in vitro data obtained in this study suggest that voriconazole may also be effective treating yeast infection in patients infected with fluconazole or itraconazole resistant isolates.

Amphotericin B↗

PubMed2005

[In vitro susceptibility of fungal and yeast clinical isolates to itraconazole and voriconazole].

The interest on the in vitro susceptibility to itraconazole has recently increased due the availability of the intravenous formulation. In this study, comparative MICs of this antifungal with voriconazole were carried out in 62 clinical isolates of filamentous fungi and 100 yeasts isolates using the NCCLS microbroth methods described in M38-A and M27-A2 documents. A MIC90 of 0.125 micrograms per ml was observed for itraconazole and voriconazole against Aspergillus fumigatus. Higher susceptibility to itraconazole was found for the filamentous form of Sporotrhix schenckii (p = 0.001). Voriconazole was more effective against Scedosporium apiospermium while Scedosporium prolificans isolates were resistant to both azoles. Some isolates of Rhizopus stolonifer were susceptible to itraconazole and resistant to voriconazole, but without statistical significance. Susceptibility of nine species of Candida was similar for both triazoles used in this study. However, Candida glabrata was more susceptible to voriconazole. Some fluconazole-resistant Candida albicans isolates were susceptible to itraconazole and / or voriconazole. Cryptococcus neoformans was more susceptible to itraconazole than to voriconazole. Itraconazole and voriconazole showed very close in vitro activity against the tested fungal isolated, except against S. schenckii. In spite of this, there were some differences in susceptibility among isolates within the same fungal species.

Antifungal Agents↗

PubMed2005

[In vitro antifungal activity of voriconazole against dermatophytes and superficial isolates of Scopulariopsis brevicaulis].

We have studied the in vitro antifungal activity of voriconazole, fluconazole and itraconazole against 252 clinical isolates of dermatophytes and Scopulariopsis brevicaulis by a standardized agar diffusion method (NeoSensitabs). Several important factors such as temperature (28 degrees C vs. 35 degrees C) and incubation time (2-10 days vs. 18-74 h) were adapted to dermatophytes and Scopulariopsis requirements. Voriconazole showed an excellent activity against most species of dermatophytes, higher than itraconazole and fluconazole. However, S. brevicaulis isolates were highly resistant to all azoles used in this study. Voriconazole might be an interesting antifungal alternative to refractory superficial mycoses.

Antifungal Agents↗

PubMed2001

Breeding of mycoparasitic Trichoderma strains for heavy metal resistance.

AIMS: This study was designed to investigate the effects of 10 heavy metals on the in vitro activities of beta-glucosidase, cellobiohydrolase, beta-xylosidase and endoxylanase enzymes for six strains of Trichoderma, and to isolate and characterize heavy metal-resistant mutants. METHODS AND RESULTS: At a concentration of 1 mmol, only mercury showed significant inhibitory effects on the in vitro enzyme activities; in all other cases, the enzymes remained active. A total of 177 heavy metal-resistant mutants were isolated and tested for cross-resistance to other heavy metals. Some mutants were effective antagonists of Fusarium, Pythium and Rhizoctonia strains, even on media containing the respective heavy metals. CONCLUSION: Trichoderma strains could be developed as biocontrol agents that are effective against plant pathogenic fungi, even under heavy metal stress. SIGNIFICANCE AND IMPACT OF THE STUDY: Trichoderma mutants resistant to heavy metals might be of value for use with heavy metal-containing pesticides, as part of an integrated plant protection system.

Drug Resistance, Fungal↗

PubMed2004

Substitution of threonine-1351 in the multidrug transporter Cdr1p of Candida albicans results in hypersusceptibility to antifungal agents and threonine-1351 is essential for synergic effects of calcineurin inhibitor FK520.

OBJECTIVES: Functional characterization of a mutant Candida albicans drug resistance protein (Cdr1p) by overexpression in Saccharomyces cerevisiae. METHODS: We overexpressed green fluorescent protein-tagged Cdr1p in S. cerevisiae AD1-8u- host and introduced a point mutation to substitute T1351 with F in Cdr1p. The cells expressing T1351F mutant Cdr1p were analysed for their functional activity using minimum inhibitory concentration, spot assay, and fluconazole efflux. The binding activity of photoaffinity analogues 8-azidoATP, iodoarylazidoprazosin and azidopine to the mutant T1351F Cdr1p was also characterized. RESULTS: The T1351F mutant Cdr1p-expressing cells were susceptible to anisomycin, cycloheximide, fluconazole, miconazole and nystatin. The mutant protein was expressed to the same level as that of native Cdr1p in S. cerevisiae cells and was properly localized to the cell surface. There was also no difference between the mutant variant and the native protein's ability to bind a photoaffinity analogue of ATP, 8-azidoATP, or the radiolabelled photoaffinity agents iodoarylazidoprazosin and azidopine. However, the substitution of T1351 resulted in considerable reduction in its ability to export fluorescent substrate rhodamine 6G. The synergy between calcineurin inhibitors FK520 and azoles was abrogated in cells expressing the T1351F mutant variant of Cdr1p. CONCLUSIONS: The results from this study suggest that the T1351 in the predicted transmembrane domain (TMD) 11 of Cdr1p is not only important for drug-substrate transport but also has a role in governing synergy of FK520.

Affinity Labels↗

PubMed2006

Identification of promoter elements responsible for the regulation of MDR1 from Candida albicans, a major facilitator transporter involved in azole resistance.

Upregulation of the MDR1 (multidrug resistance 1) gene is involved in the development of resistance to antifungal agents in clinical isolates of the pathogen Candida albicans. To better understand the molecular mechanisms underlying the phenomenon, the cis-acting regulatory elements present in the MDR1 promoter were characterized using a beta-galactosidase reporter system. In an azole-susceptible strain, transcription of this reporter is transiently upregulated in response to either benomyl or H(2)O(2), whereas its expression is constitutively high in an azole-resistant strain (FR2). Two cis-acting regulatory elements within the MDR1 promoter were identified that are necessary and sufficient to confer the same transcriptional responses on a heterologous promoter (CDR2). One, a benomyl response element (BRE), is situated at position -296 to -260 with respect to the ATG start codon. It is required for benomyl-dependent MDR1 upregulation and is also necessary for constitutive high expression of MDR1. A second element, termed H(2)O(2) response element (HRE), is situated at position -561 to -520. The HRE is required for H(2)O(2)-dependent MDR1 upregulation, but dispensable for constitutive high expression. Two potential binding sites (TTAG/CTAA) for the bZip transcription factor Cap1p (Candida AP-1 protein) lie within the HRE. Moreover, inactivation of CAP1 abolished the transient response to H(2)O(2). Cap1p, which has been previously implicated in cellular responses to oxidative stress, may thus play a trans-acting and positive regulatory role in the H(2)O(2)-dependent transcription of MDR1. A minimal BRE (-290 to -273) that is sufficient to detect in vitro sequence-specific binding of protein complexes in crude extracts prepared from C. albicans was also defined. Interestingly, the sequence includes a perfect match to the consensus binding sequence of Mcm1p, raising the possibility that MDR1 may be a direct target of this MADS box transcriptional activator. In conclusion, while the identity of the trans-acting factors that bind to the BRE and HRE remains to be confirmed, the tools developed during this characterization of the cis-acting elements of the MDR1 promoter should now serve to elucidate the nature of the components that modulate its activity.

Antifungal Agents↗